The basic studies described in project one are focused on mechanistic studies of autoimmunity, self-tolerance, and immune modulation in human CD4 T cells. Recent studies have identified a gene product GRAIL, required for maintenance of unresponsiveness in murine and human CD4 T cells. When GRAIL is present, both murine and human CD4 T cells have a markedly decreased ability to proliferate in response to activation signals. Mechanism of action studies for three therapeutic agents developed for clinical intervention in autoimmunity and transplantation (Rapamycin, anti-IL-2, and CTLA-4Ig). have linked molecular regulators of CD4 T-cell anergy including GRAIL and its epistatic regulators, to therapeutic effect These studies demonstrated that activation of naive, but not memory CD4 T cells, could be blocked by treatment with all three drugs and that the therapeutic effect was linked to the selective inhibition of the translation of an epistatic regulator of GRAIL, called Otubain-1 (Otub-1), that maintained nai've CD4 T cell unresponsiveness (anergy). Preliminary studies suggest that these findings translate in human CD4 T cells as well. A second set of preliminary data were generated in the study of murine regulatory T cells (Tregs), where obligate cellxell interaction requirements between Tregs (surface CTLA4) and T effectors (Teffs) (TCR induced B7 molecules) were characterized, that can be translated in human CD4 Tregs and Teffs as described below. Four Specific Aims have been developed to address the following hypotheses: (1) naive and memory human CD4 T cells are held in check by GRAIL expression and overcoming GRAIL mediated inhibition of activation is differentially regulated in naive versus memory human CD4 T cells, thus requiring selective agents for therapeutic effect; and (2) CD4 Tregs regulate CD4 Teffs through Treg CTLA4 engagement of activation induced B7 products on Teffs, (3) expression of GRAIL and its epistatic regulators (in Tregs or Teffs) may be important in certain autoimmune diseases, and, (4) there may be a defect in Teffs, not Tregs, in autoimmune disease. The significance of these studies in human T cells is the potential for characterization of mechnism of action of immune inteventions to allow appropriate CD4 T cell targeted therapy.